Predicting Response to Anti-CD40 Therapy in DLBCL Patients

ABSTRACT

This invention provides methods, compositions, and kits relating to biomarkers whose expression levels are correlated with diffuse large B-cell lymphoma (DLCBL) patients&#39; response to treatment with a CD20 antagonist, such as a CD20 antibody, exemplified by rituximab. The methods, compositions, and kits of the invention can be used to identify DLBCL patients who are likely or not likely, to respond to anti-CD20 treatments.

BACKGROUND OF THE INVENTION

Lymphoma is the fifth most common cancer in women and the sixth most common cancer in men in the Western world, see Murawski et al. (2010) Unresolved issues in diffuse large B-cell lymphomas, Expert Rev. Anticancer Ther. 10(3):387. 90% of aggressive lymphomas originate from B-cells and are classified as diffuse large B-cell lymphomas (DLBCL). Until recently, the accepted form of therapy for DLBCL was CHOP: a combination of cyclophosphamide, hydroxydaunorubicine (doxorubicin), Oncovin® (vincristine) and prednisone. In 1997, the FDA approved rituximab (Rituxan®) for treatment of aggressive Non-Hodgkin lymphomas. Rituximab is a chimeric mouse-human monoclonal antibody against a protein CD20 found primarily on the surface of B-cells. Rituximab has been shown to be effective as a single agent in DLBCL, Coiffier et al. (1998) Rituximab (anti-CD20 monoclonal antibody) for the treatment of patients with relapsing or refractory aggressive lymphoma: a multicenter Phase II study. Blood 92(6):1927. Subsequent studies of a combination of rituximab with CHOP demonstrated high response rate with high overall and progression-free survival, even with fewer rounds of CHOP therapy, Murawski et al. (2010), supra. However, it appears that patients receiving rituximab have different response rates, which result in different survival times. Thus, there is a need to identify patients who would benefit from the anti-CD 20 therapy such as rituximab treatment and patients who would likely not respond well to such therapy, and would need a different therapy instead.

SUMMARY OF THE INVENTION

The invention is a method of predicting whether a patient with diffuse large B-cell lymphoma (DLBCL) is likely to exhibit response to anti-CD20 therapy, comprising: obtaining a sample from the patient; determining in the sample the level of expression of one or more genes listed in Table 1 and Table 2; determining whether the patient is likely to exhibit response to anti-CD20 therapy, based on the similarity between the measured level of expression of each gene in the patient's sample and a set of control samples. In some variations of this embodiment, the anti-CD20 therapy comprises an anti-CD20 antibody, for example rituximab.

In another embodiment, the invention is a method of treatment of a diffuse large B-cell lymphoma (DLBCL) patient, comprising: obtaining a sample from the patient; detecting in the sample the expression of one or more genes listed in Table 1 and Table 2; determining whether the patient is likely to exhibit response to anti-CD20 therapy, based on the similarity between the measured expression of each gene measured in the patient's sample and a set of control samples; and administering rituximab if the patient is determined to be likely to respond to anti-CD20 therapy.

In another embodiment, the invention is a set of diagnostic probes for predicting whether a patient with diffuse large B-cell lymphoma (DLBCL) is likely to exhibit response to anti-CD20 therapy, comprising nucleic acid probes or antibodies for detecting expression of one or more genes listed in Table 1 and Table 2.

In another embodiment, the invention is a kit for predicting whether a patient with diffuse large B-cell lymphoma (DLBCL) is likely to exhibit response to anti-CD20 therapy comprising a set of nucleic acid probes for one or more genes listed in Table 1 and Table 2; and reagents necessary for detecting hybridization of the nucleic acid probes. In variations of this embodiment, the kit further comprises antibodies for proteins expressed from the genes listed in Table 1 and Table 2.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a Kaplan-Meier plot showing differences in survival for the model built in Example I validated with re-substitution.

FIG. 2 is a Kaplan-Meier plot showing differences in survival for leave-one-out cross validation of the model built in Example 1.

FIG. 3 is a Kaplan-Meier plot constructed with the classification according to the SVM method for the model built in Example 2.

FIG. 4 is a Kaplan-Meier plot constructed with the probability assigned according to the SVM method for the model built in Example 2.

FIG. 5 is a Kaplan-Meier plot showing differences in survival for the independent set of samples in Example 3.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The terms “array,” “microarray,” and “DNA chip” are used herein interchangeably to refer to an array of distinct polynucleotides affixed to a substrate, such as glass, plastic, paper, nylon, or other type of membrane, filter, chip, or any other suitable solid support. The polynucleotides can be synthesized directly on the substrate, or synthesized separate from the substrate and then affixed to the substrate. Commercial arrays containing probes for all the targets potentially present in a particular type of sample are available. A commercial array may contain probes for all the nucleotide sequences present in the genome. Alternatively, an array may contain probes for only expressed sequences. The term “custom array” refers to an array that contains probes for only selected targets. In the context of the present invention, a custom array may contain probes for some or all of the genes in the expression profile predictive of response to anti-CD20 therapy.

The term “biomarker” refers to a gene or nucleic acid sequence that is of interest for a particular phenotype, such as a disease. For example, a biomarker could be a protein-coding gene whose activation or inactivation leads to a disease. In that case, the presence of the mRNA or the protein could be indicative or predictive of the disease. In another example, a biomarker could be a sequence polymorphism in linkage disequilibrium with an unknown gene causing disease. In that case, the presence of the polymorphism could be indicative or predictive of the disease. In yet another example, a biomarker is a somatic mutation in a gene, wherein the presence of the mutation is correlated with a disease. In that case, the presence of the mRNA mutation could be indicative or predictive of the disease. In the context of the present invention, biomarkers are the genes whose expression (measured by the presence of the mRNA or protein) is in correlation with response or lack of response to anti-CD20 therapy.

The terms “gene expression profile” or “gene expression signature” refer to a collection of expression levels of a number of genes. The genes in the profile are markers that discriminate between individuals with different phenotypes. The discrimination is achieved because the expression levels of each gene exhibit a statistically significant difference among groups of individuals with different phenotypes. Thus each of the phenotypes is characterized by a lowered expression of some genes in the profile, and overexpression of other genes in the profile. By determining the “expression profile,” i.e. expression of some or all the genes in the profile, one can assign a phenotype to an individual whose phenotype has not yet manifested itself. In the context of the present invention, by determining the gene expression profile disclosed herein, one can predict a DLBCL patient's response to anti-CD20 therapy.

The term “hybridization” refers to the formation of a duplex between two single-stranded nucleic acids due to complementary base pairing. Hybridization can occur between perfectly complementary nucleic acid strands or between substantially (but not perfectly) complementary nucleic acid strands that contain one or more mismatches. Conditions under which only perfectly complementary nucleic acid strands will hybridize are referred to as “stringent” hybridization conditions. Stable duplexes of substantially complementary nucleic acids can be achieved under less stringent hybridization conditions. Those skilled in the art of nucleic acid technology can determine duplex stability using for example, computer software such as Visual OMP® (DNA Software, Inc., Ann Arbor, Mich.).

The term “primer” refers to an oligonucleotide that acts as a point of initiation of DNA synthesis under suitable conditions in the presence of nucleic acid precursors and an agent for polymerization. A primer can either consist entirely of the target-hybridizing region or can contain additional features which allow for detection, immobilization, or manipulation of the amplified product.

The term “probe” refers to a nucleic acid that selectively hybridizes to a target nucleic acid under suitable conditions. A probe can either consist entirely of the target-hybridizing region or can contain additional features which allow for the detection, immobilization, or manipulation of the probe-target duplex. The probe may contain modifications to its primary structure by the addition of labels, linkers, peptides or any other groups necessary to perform the detection assay in the chosen format.

The term “probe set” refers to a unique set of oligonucleotides in a microarray capable of detecting a marker. Typically, a microarray contains several probe sets for detecting each marker or gene. In commercial microarrays, each probe set is associated with a sequence that is searchable in a public database by a unique accession number.

The terms “target sequence” or “target” refer to a region of a nucleic acid that is to be analyzed.

The terms “nucleic acid,” “polynucleotide” and “oligonucleotide” refer to target sequences, primers and probes. The terms are not limited by length and are generic to linear polymers of deoxyribonucleotides (single-stranded or double-stranded DNA), ribonucleotides (RNA), and any other N-glycoside of a purine or pyrimidine base, including adenosine, guanosine, cytidine, thymidine and uridine and modifications of these bases.

The term “response to therapy” refers to a benefit attributable to therapy. Response may be assessed by measuring a clinically relevant parameter, such as tumor shrinkage, length of overall survival or survival without disease progression. For example, the clinically relevant parameter may be the length of survival following the start of the therapy. The patient who has survived for a certain time, e.g. at least three years, may be considered to have responded to therapy. The patient, who has not survived past three years, is considered to not have responded or responded poorly to therapy.

The term “sample” refers to any composition containing or presumed to contain nucleic acid. This includes a sample of tissue or fluid isolated from an individual for example, skin, plasma, serum, spinal fluid, lymph fluid, synovial fluid, urine, tears, blood cells, organs and tumors, including the fresh-frozen tissue and formalin-fixed paraffin embedded tissue (FFPET), and also to samples of in vitro cultures established from cells taken from an individual, and nucleic acids isolated therefrom.

The term “training set” or “control set” refers to a set of samples used to establish a correlation between two variables. For example, a training set is a set of patient samples used to establish a correlation between the expression of a gene and the patient's condition. In the context of the present invention, the training set is a set of samples from DLBCL patients receiving anti-CD20 therapy, for whom survival information is available. The training set is used to establish a correlation between the expression profile and survival.

The term “testing set” refers to a set of samples used to verify the correlation established using the training set. For example, a testing set is a set of samples from patients for whom both gene expression and the patient's condition are known. This set is used to verify whether the correlation established using the training set will correctly predict the patient's condition. In the context of the present invention, the testing set is an independent set of samples from DLBCL patients receiving anti-CD20 therapy, for whom survival information is available. The testing set is used to measure expression of the genes in the profile and test whether the actual survival data matches the prediction made by measuring the gene expression.

The term “test sample” refers to a patient's sample that is to be tested for a particular parameter.

The present invention is based on the discovery that expression of certain genes predicts response of diffuse large B-cell lymphoma (DLBCL) patients to anti-CD20 therapy. Specifically, it was discovered that some genes exhibit differential expression between a group of DLBCL patients with response to rituximab therapy and patients with poor response to rituximab therapy, where the response is measured by the length of survival following the therapy. The invention comprises such genes and gene expression profiles containing such genes. The invention further comprises the use of the genes and gene expression profiles to predict response of DLBCL patients to anti-CD20 therapy.

The genes identified as differentially expressed in responders and poor responders to anti-CD20 therapy among DLBCL patients are listed in Tables 1 and 2. In Tables 1 and 2, the column “PS ID” lists the identification code for the probe set in the Affymetrix GeneChip® Human Genome U133 Plus 2.0 Array (Affymetrix, Santa Clara, Calif.) The column “NCBI” lists the corresponding public reference number (NCBI Accession No.). The column “Gene Title” lists the names of the genes, where available. The column “Gene Symbol” lists the gene symbols, where available. The column “p-value” in Table 1 lists the p-value statistic. The column “FDR_BH” in Table 2 lists the values of the statistic False Discovery Rate (FDR), calculated according to the method described in Benjamini Y. and Hochberg, Y., (1995) J Royal Stat. Soc. Ser. B 57:289.

TABLE 1 Predictive markers identified in Example 1 PS ID NCBI Symbol Gene Title p−value 202858_at NM_006758 U2AF1 U2 small nuclear RNA auxiliary factor 1 8.40E−05 205215_at NM_007212 RNF2 ring finger protein 2 4.10E−05 205877_s_at NM_017590 ZC3H7B zinc finger CCCH-type containing 7B 4.10E−05 208656_s_at AF135162 CCNI cyclin I 1.90E−05 208776_at BF432873 PSMD11 proteasome (prosome, macropain) 6.00E−05 26S subunit, non-ATPase, 11 210461_s_at BC002448 ABLIM1 actin binding LIM protein 1 3.50E−05 210964_s_at U94364 GYG2 glycogenin 2 6.20E−05 212669_at AI093569 CAMK2G calcium/calmodulin-dependent 6.70E−06 protein kinase (CaM kinase) II gamma 212678_at AW054826 NF1 neurofibromin 1 (neurofibromatosis, 3.40E−05 von Recklinghausen disease, Watson disease) 213658_at BE858194 — mRNA full length insert cDNA 3.70E−08 clone EUROIMAGE 826033 213748_at AW271713 TRIM66 tripartite motif-containing 66 3.30E−05 214891_at U79257 FBXO21 F-box protein 21 4.90E−05 218205_s_at NM_017572 MKNK2 MAP kinase interacting serine/threonine kinase 2 6.50E−05 224642_at BG291550 FYTTD1 forty-two-three domain containing 1 7.70E−05 224808_s_at AI090768 GET4 Golgi to ER traffic protein 4 homolog 7.80E−05 226004_at AI910855 CABLES2 Cdk5 and Abl enzyme substrate 2 3.60E−05 227844_at AI089932 FMNL3 formin-like 3 2.50E−05 230566_at AI806805 C22orf27 chromosome 22 open reading frame 27 6.70E−05 231997_at R69910 TBCEL tubulin folding cofactor E-like 6.90E−05 232076_at AW294133 ZNF707 zinc finger protein 707 3.60E−05 235640_at AI763196 — transcribed locus 7.80E−05 236449_at AI885390 CSTB cystatin B (stefin B) 2.70E−05 236604_at BF195603 BAHCC1 BAH domain and coiled-coil containing 1 2.00E−05 239656_at AA827176 LOC723809 hypothetical LOC723809 6.90E−05 240377_at AI344289 NPIP nuclear pore complex interacting protein 3.40E−05 241388_at AL567118 — cDNA FLJ40566 fis, clone THYMU2004733 1.40E−05 243518_at BF195694 LOC730367 hypothetical protein LOC730367 1.10E−05 1553326_at AF453828 RXFP2 relaxin/insulin-like family peptide receptor 2 3.70E−05 1554732_at BC020886 MGC24125 hypothetical protein MGC24125 2.40E−05 1566337_x_at AJ293390 — mRNA, differentially expressed in 5.80E−05 malignant melanoma, clone MM A2

TABLE 2 Predictive markers identified in Example 2 PS ID NCBI Gene Title Symbol FDR_BH 200730_s_at BF576710 protein tyrosine phosphatase type IVA, PTP4A1 1.79E−07 member 1 201800_s_at AF185696 oxysterol binding protein OSBP 1.79E−07 213359_at W74620 heterogeneous nuclear ribonucleo- HNRNPD 1.79E−07 protein D (AU-rich element RNA binding protein 1, 37 kDa) 200848_at AA479488 adenosylhomocysteinase-like 1 AHCYL1 2.45E−07 37170_at AB015331 BMP2 inducible kinase BMP2K 3.00E−07 202438_x_at BF346014 iduronate 2-sulfatase IDS 6.15E−07 242814_at AI986192 serpin peptidase inhibitor, clade B SERPINB9 6.15E−07 (ovalbumin), member 9 233396_s_at AK023759 CSRP2 binding protein CSRP2BP 8.81E−07 211744_s_at BC005930 CD58 molecule CD58 8.98E−07 233509_at AK021844 hect domain and RLD 4 HERC4 8.98E−07 210093_s_at AF067173 mago-nashi homolog, proliferation- MAGOH 1.24E−06 associated (Drosophila) 223892_s_at AF182414 transmembrane BAX inhibitor motif TMBIM4 1.60E−06 containing 4 243361_at N51597 splicing factor, arginine/serine-rich 12 SFRS12 2.02E−06 216252_x_at Z70519 Fas (TNF receptor superfamily, FAS 2.68E−06 member 6) 231369_at BG149482 Zinc finger protein 333 ZNF333 2.68E−06 243267_x_at AI127295 — — 2.79E−06 1555063_at BC029495 ubiquitin specific peptidase 6 (Tre-2 USP6 3.39E−06 oncogene) 225026_at BF572029 chromodomain helicase DNA binding protein 6 CHD6 4.47E−06 236246_x_at BF195670 hypothetical protein LOC653160 LOC653160 4.47E−06 24393l_at R64696 — — 4.47E−06 200918_s_at NM 003139 signal recognition particle receptor SRPR 5.19E−06 (docking protein) 215719_x_at X83493 Fas (TNF receptor superfamily, FAS 5.33E−06 member 6) 224917_at BF674052 microRNA 21 MIR21 5.33E−06 225173_at BE501862 Rho GTPase activating protein 18 ARHGAP18 5.33E−06 239387_at AW004885 — — 5.33E−06 238549_at AI420611 core-binding factor, runt domain, alpha CBFA2T2 5.77E−06 subunit 2; translocated to 2 201150_s_at NM 000362 TIMP metallopeptidase inhibitor 3 TIMP3 6.02E−06 218924_s_at NM004388 di-N-acetyl-chitobiase CTBS 6.92E−06 228822_s_at AI435036 ubiquitin specific peptidase 16 USP16 7.26E−06 210621_s_at M23612 RAS p21 protein activator (GTPase RASA1 7.69E−06 activating protein) 1 229961_x_at AI871270 YjeF N-terminal domain YJEFN3 8.19E−06 containing -3

Measuring expression of one or more of the genes in the profile allows predicting whether a DLBCL patient is, or is not likely to respond to anti-CD20 therapy, such as for example, rituximab. In the context of the present invention, measuring the level of expression of a gene in a patient's sample may be accomplished by several means. Gene expression may eb measured by measuring the level of RNA expressed by the gene. The amount of RNA may be determined for example, by reverse transcription followed by quantitative PCR (qPCR) amplification. Gene expression may also be detected by hybridizing a single probe to the RNA in a blot or hybridizing the RNA to multiple probes in an array and quantifying the hybridization signal. Alternatively, expression of protein-coding gene can be determined by measuring the level of expression of the protein encoded by the gene or measuring enzymatic activity of the expressed protein consisting in part or entirely of the protein encoded by the gene. As an additional alternative, it is possible to indirectly assess the expression of a gene listed in Table 1 or Table 2 by measuring the level of expression or activity of a gene or protein situated downstream in a biological pathway from any gene listed in Table 1 or Table 2. For such downstream pathway gene or protein, the expression may also be measured at various levels, including the nucleic acid level, protein level, and activity level.

To predict likelihood of a DLBCL patient's response to anti-CD20 therapy, expression of at least one, more than one or all of the genes listed in Tables 1 and 2 may be measured. A person skilled in the art of statistics would recognize that a smaller subset of genes among those listed in Tables 1 and 2 may be sufficient to predict a patient's response. For example, a subset of genes may be selected based on the value of the T-statistic, the p-value or the FDR value. A person skilled in the art is aware of the statistical methods for selecting a subset of genes from an expression profile. For example, the smallest number of genes that is sufficient to predict response may be determined by calculating the area under the curve of the receiver operating characteristic (“AUC of RUC” method) as described in Green, D. M., Swets, J. A., (1966) Signal detection theory and psychophysics, Wiley, N.Y.; Swets, J. A., Pickett, R. M. (1982) Evaluation of diagnostic systems: Methods from signal detection theory, Academic Press, New York; and Pepe, M. S. (2003), The statistical evaluation of medical tests for classification and prediction, Oxford Univ. Press.

Additional genes may be added to the profile predictive of a DLBCL patient's response to anti-CD20 therapy, as long as expression of the genes differs between the patients who respond and patients who do not respond to anti-CD20 therapy. For example, when measuring the difference in expression, the p-value or the FDR value may be used to select additional genes to be added to the profile.

In the examples of the expression profiles of the present invention, the expression of each gene was measured in a first set of patient samples whose response (or lack of response) to anti-CD20 therapy has been documented. This first set of samples (referred to as the training set) contained an adequate number of responder patients and an adequate number of non-responder patients.

To predict response to anti-CD20 therapy in a patient, the expression profile in the patient's sample was determined and compared to the expression profile in the samples of the training set. A statistical model was used to determine whether based on the expression profile, the patient is likely to belong to a responder group or a non-responder group for the anti-CD20 therapy. In the method of the present invention, the gene expression in the test sample and the samples of the training set need not be measured simultaneously. For convenience, expression data from the training set samples may be stored on a computer readable medium and accessed each time a new patient sample is tested.

Response Prediction

In one embodiment, the invention comprises a method of predicting response of a

DLBCL patient to anti-CD20 therapy, such as rituximab. The method comprises obtaining a sample from a patient diagnosed with DLBCL and measuring expression level of one or more genes listed in Tables 1 and 2 prior to the patient receiving the therapy and comparing the expression to the expression of the same genes in the samples of the training set.

The expression levels of any gene listed in Tables 1 and 2 may be measured for example, by measuring the level of mRNA expressed from the gene in the patient's sample by reverse transcription and quantitative PCR (qPCR) amplification of the resulting cDNA, see U.S. Pat. Nos. 5,210,015 and 5,487,972 and Holland et al. (1991) PNAS USA 88: 7276. For multiple genes, the levels of mRNA may be measured in a microarray-based assay. For example, multiple mRNA molecules can be reverse-transcribed using common poly-A primers, converted into cDNA and then into labeled amplified RNA (aRNA) that is applied to an array of immobilized probes. (MAQC Consortium (2006) Nat Biotechnol. 24(9):1151-61). The expression may be detected using commercially available microarrays, for example from Roche NimbleGen, Inc. (Madison, Wisc.) or Affymetrix, Inc. (Santa Clara, Calif.). Alternatively, only selected probe sets for each of the genes listed in Tables 1 and 2 may be used. The sequences of probes corresponding to each gene are listed in the literature accompanying the Affymetrix GeneChip® Human Genome U133 Plus 2.0 Array product. A person skilled in the art of nucleic acid hybridization may also design custom hybridization probes for the genes listed in Tables 1 and 2 using the gene sequences available through the public databases. A person skilled in the art of nucleic acid arrays may also design a custom array containing previously known or custom designed probes for one or more, or all the genes listed in Tables 1 and 2 according to DNA Microarrays: A Molecular Cloning Manual, (2003), Eds. Bowtell and Sambrook, Cold Spring Harbor Laboratory Press.

Any alternative methods of amplifying and quantifying mRNA or cDNA may also be used in the context of the present invention. Furthermore, as an alternative to detecting gene transcripts on the nucleic acid level, the protein products of the genes in Tables 1 and 2 may be detected, where the protein products are present. The proteins may be detected, for example, by immunoassays. In another variation of this embodiment, the mRNA transcripts or protein products of genes that are linked to the genes in Tables 1 and 2 by a biological pathway, may be detected instead of (or in addition to) detecting the mRNA transcripts or proteins corresponding to the genes in Tables 1 and 2.

After the expression level of one or more genes from Tables 1 and 2 has been determined in the patient's sample, the expression is compared to the documented expression of the same gene or genes in the set of samples taken from patients whose response anti-CD20 therapy (e.g. rituximab) has been documented (training set). A statistical tool is then used to determine whether the test patient will respond to anti-CD20 therapy such as rituximab. For example, to determine whether the patient is classified in the responder group or a non-responder group, the Support Vector Machine (SVM) method (Cortes, C. and Vapnik, V. (1995) Machine Learning, 20:273-297) or the k-nearest neighbor method described e.g. in Hastie et al., (2001) The Elements of Statistical Learning: Data Mining, Inference, and Prediction, Springer, N.Y., may be used.

Method of Treatment

In another embodiment, the invention is a method of treatment of DLBCL. The method comprises collecting a sample from a patient diagnosed with DLBCL and measuring the level of expression of one or more genes from Tables 1 and 2 in the sample. In variations of this embodiment, mRNA levels or levels of the protein products of the genes from Tables 1 and 2 may be detected. In another variation of this embodiment, the transcripts or protein products of genes that are linked to the genes in Tables 1 and 2 by a biological pathway may be detected instead of (or in addition to) detecting the transcripts or proteins corresponding to the genes in Tables 1 and 2.

After the expression level of one or more genes from Tables 1 and 2 has been determined in the patient's sample, the expression is compared to the expression of the same gene or genes in samples of patients whose response to anti-CD20 therapy has been documented. A statistical tool is then used to determine whether the test patient will likely respond to anti-CD20 therapy. If the patient is predicted to respond to anti-CD20 therapy, the therapy (e.g. an antibody such as rituximab) is administered alone or in combination with additional therapeutic agents.

Gene Expression Profile

In another embodiment, the invention comprises a set of detection probes for predicting response of DLBCL patients to anti-CD20 therapy (e.g. rituximab). The detection probes may comprise nucleic acid probes for detecting expression of the genes listed in Table 1, or Table 2, or both. The exact sequence of each nucleic acid probe is not critical. A person skilled in the art of nucleic acid hybridization will be able to select a probe or probes for detecting expression of each gene based on the published sequence, and assemble the set of probes according to the present invention. For example, for each gene listed in Table 1 or Table 2, the probes listed in the product literature for the Affymetrix GeneChip® Human Genome U133 Plus 2.0 Array may be used to assemble the set of probes according to the present invention.

In some embodiments, detection probes other than nucleic acid probes may be used. For example, antibodies may be used to detect the presence of proteins that are encoded by the genes listed in Table 1 or Table 2, where such proteins are available. In some embodiments, probes or antibodies for the genes linked to the genes listed in Table 1 or Table 2 in a biological pathway may also be included. Thus in some embodiments of the invention, the set of detection probes includes nucleic acid probes, antibodies, or a combination of both.

Kits

In another embodiment, the invention is a kit for predicting response to anti-CD20 therapy in a DLBCL patient. An exemplary kit comprises probes that detect expression of at least one, two or all of the genes listed in Tables 1 and 2. The kit may comprise additional reagents necessary to extract and amplify RNA from the patient's sample. In variations of this embodiment, the kit comprises a set of oligonucleotide probes that detect expression of at least one, two or all of the genes set forth in Tables 1 and 2, attached to a solid support, such as a microarray slide or chip. The kit may also contain reference material, e.g. samples from patients whose response to anti-CD20 therapy, for example, rituximab, has been documented.

In some embodiments of the invention, the kit may contain antibodies for detecting proteins that are encoded by the genes set forth in Tables 1 and 2. In some embodiments, probes or antibodies for the genes linked to the genes listed in Table 1 or Table 2 in a biological pathway may also be included. The kit may comprise additional reagents for antibody-based detection of proteins.

EXAMPLES Example 1 Selecting Predictive Expression Markers in DLBCL Patients Treated with Rituximab Using the Cox Proportional Hazards Model and a K-Nearest-Neighbor Method

Samples were obtained from DLBCL patients undergoing therapy with CHOP-R (CHOP in combination with rituximab). For these patients, the three-year survival status has been documented. The samples underwent gene expression profiling through use of an Affymetrix GeneChip® Human Genome U133 Plus 2.0 Array (Affymetrix, Inc. Santa Clara, Calif.). Applying a quality control, samples with low signals or less than 18% present calls (microarray signals) were eliminated. The resulting pool consisted of 85 patients falling into two groups: Group 1-21 long-term survivors, with survival greater than three years (1095 days) and Group 2-19 short term survivors, who died prior the end of three years and 45 patients who dropped out prior to the end of three years.

The microarray expression data was analyzed using the Cox proportional hazards model (Cox, D. R. (1972) Regression models and life tables, J R Statistical Soc. Ser., 34:187-220, Lachin, J. M. (2000) Biostatistical Methods: The Assessment of Relative Risks, Wiley, N.Y.). For each gene, the Cox model was used to test association between the level of expression of the gene and survival past three years. Based on the value of the T-statistic, the Cox model selected 200 genes. Of the 200 genes, 30 genes with the lowest p-value (Table 1) were selected for the model.

Next, the 1-nearest neighbor method (a version of the k-nearest neighbor method with k=1 (T. Hastie et al., (2001) The Elements of Statistical Learning: Data Mining, Inference, and Prediction, Springer, N.Y.)), was used to group patients according to the gene expression. The model was tested with two methods of cross-validation.

The first validation was performed using the re-substitution method. The accuracy of re-substitution validation was 90%. Results of the re-substitution validation are shown on FIG. 1. FIG. 1 is a Kaplan-Meier plot (Kaplan, E. L. and Meier, P. (1958) J. Amer. Stat. Assn. 53:457) demonstrating the difference in survival between the two groups selected according to the model. The upper curve represents the actual survival data for patients predicted to be respondents, while the lower curve represents the actual survival data for patients predicted to be non-respondents by the method of the present invention.

The second validation was performed using the leave-one-out cross validation method. The accuracy of leave-one-out cross-validation was 72%. Results of the leave-one-out cross validation are shown on FIG. 2. FIG. 2 is a Kaplan-Meier plot demonstrating the difference in survival between the two groups selected according to the model. The upper curve represents the actual survival data for patients predicted to be respondents, while the lower curve represents the actual survival data for patients predicted to be non-respondents by the method of the present invention.

Example 2

Selecting Predictive Expression Markers in DLBCL Patients Treated with Rituximab Using the Cox Proportional Hazards Model and Support Vector Machine Method

Samples were obtained from DLBCL patients undergoing therapy with CHOP and patients undergoing CHOP-R (CHOP in combination with rituximab) therapy. 159 patients for whom the three-year survival status has been documented were selected. Samples from patients who left the study prior to the end of the three year period were not used in this example.

The 159 samples were randomly split into a training and a testing set in three separate runs as follows: Run 1: 75 training, 84 testing; Run 2: 78 training, 81 testing; Run 3: 83 training, 76 testing. The samples underwent gene expression profiling through use of an Affymetrix GeneChip® Human Genome U133 Plus 2.0 Array.

The microarray expression data was analyzed using the Cox proportional hazards model. For each gene, the Cox model was used to test association between the level of expression and survival past three years. The Cox model was applied to the three testing sets (Runs 1, 2 and 3). The genes with the lowest False Discovery Rate (FDR) were selected. The FDR cut-off of 0.01 yielded 31 genes (Table 2). These 31 genes were selected for model building and cross-validation.

To predict patient's survival past three years, the Support Vector Machine (SVM) method (Cortes, C. and Vapnik, V. (1995) Machine Learning, 20:273-297) was used. SVM is a binary classifier that classifies an input into one of the two classes based on the input data. SVM was applied to classify patient samples into Group 1 (survival more than three years) or Group 2 (survival less than three years) based on the expression levels of the 31 genes in Table 2.

After the samples were separated into Group 1 and Group 2 using the SVM method, a Kaplan-Meier plot was assembled (FIG. 3). Group 1 contained 51 samples and Group 2 contained 33 samples. The upper curve represents the actual survival data for patients predicted to be respondents, while the lower curve represents the actual survival data for patients predicted to be non-respondents by the method of the present invention. The Log Rank test was used to determine whether the two curves (for Group 1 and Group 2) were significantly different. Log Rank test yielded a highly significant p- value of 6.06×10⁻¹⁰.

Alternatively, SVM was used to determine the probability that a sample belongs to one of the two groups. This approach yields three classifications: Group 1, Group 2 and unclassified samples for whom the probability of belonging to either group is less than 90%. After the samples were separated into Group 1 and Group 2 using the SVM method, a Kaplan-Meier plot was assembled (FIG. 4). Group 1 contained 27 samples, Group 2 contained 31 samples and the remaining 26 samples were unclassified. Log Rank test yielded a highly significant p-value of 4.54×10⁻¹¹. FIG. 4 shows the Kaplan-Meier plot for the 84 samples from the testing set in Run 1. The upper curve represents the actual survival data for patients predicted to be respondents, while the lower curve represents the actual survival data for patients predicted to be non-respondents by the method of the present invention. The middle curve represents the actual survival data for the unclassified group.

Example 3 Validating the Model on DLBCL Patients Treated with Rituximab

The method was applied to an independent set of samples: 233 samples from DLBCL patients on the CHOP-R therapy for whom survival data was available. SVM was used to determine the probability of each patient belonging to Group 1 (survival more than three years) or Group 2 (survival less than three years). Samples with low probability of belonging to either group remained unclassified. After the samples were separated into Group 1 and Group 2 using the SVM method, a Kaplan-Meier plot was assembled using the survival information available for each sample (FIG. 5). The middle curve represents the actual survival data for patients predicted to be respondents, while the lower curve represents the actual survival data for the unclassified patients. The upper curve represents the actual survival data for patients predicted to be non-respondents by the method of the present invention. The data does not contradict the teachings of the invention. The upper curve represents only three patients, all of whom left the study prior to the expiration of the three-year period (at 1.71, 2.95 and 5.37 years). Unfortunately, although the curve shows a 100% survival, the censored patients may have passed away within the three-year period.

While the invention has been described in detail with reference to specific examples, it will be apparent to one skilled in the art that various modifications can be made within the scope of this invention. Thus the scope of the invention should not be limited by the examples described herein, but by the claims presented below. 

1. A method of predicting whether a patient with diffuse large B-cell lymphoma (DLBCL) is likely to exhibit response to anti-CD20 therapy, comprising: (a) obtaining a sample from the patient; (b) determining in the sample the level of expression of one or more genes selected from U2AF1, RNF2, ZC3H7B, CCNI, PSMD11, ABLIM1, GYG2, CAMK2G, NF1, gene coding for cDNA clone EUROIMAGE 826033 (NCBI Accession No. BE858194), TRIM66, FBXO21, MKNK2, FYTTD1, GET4, CABLES2, FMNL3, C22orf27, TBCEL, ZNF707, the gene corresponding to NCBI Accession No. AI763196, CSTB, BAHCC1, LOC723809, NPIP, the gene coding for CDNA FLJ40566 fis (NCBI Accession No. AL576118), LOC730367, RXFP2, MGC24125, the gene coding for mRNA, differentially expressed in malignant melanoma, clone MM A2 (NCBI Accession No. AJ293390), PTP4A1, OSBP, HNRNPD, AHCYL1, BMP2K, IDS, SERPINB9, CSRP2BP, CD58, HERC4, MAGOH, TMBIM4, SFRS12, FAS, ZNF333, the gene corresponding to NCBI Accession No. AI127295, USP6, CHD6, LOC653160, the gene corresponding to NCBI Accession No. R64696, SRPR, MIR21, ARHGAP18, the gene corresponding to NCBI Accession No. AW004885, CBFA2T2, TIMP3, CTBS, USP16, RASA1 and YJEFN3; (c) determining whether the patient is likely to exhibit response to anti-CD20 therapy, based on the similarity between the level of expression of each gene measured in step (b) in the patient's sample and a set of control samples, where the expression of the corresponding genes has been determined.
 2. The method of claim 1, wherein anti-CD20 therapy comprises an anti-CD20 antibody.
 3. The method of claim 1, wherein the anti-CD20 antibody is rituximab.
 4. The method of claim 1, wherein in step (b), the level of expression of all the genes listed in Table 1 is determined.
 5. The method of claim 1, wherein in step (b), the level of expression of all the genes listed in Table 2 is determined.
 6. The method of claim 1, wherein the expression of the genes is measured by measuring the level of RNA transcribed from each gene.
 7. The method of claim 1, wherein the expression of the genes is measured by measuring the level of protein corresponding to each gene.
 8. (canceled)
 9. The method of claim 1, wherein the expression of the genes is measured by measuring the product of activity of a protein corresponding to a gene listed in Table 1 or Table
 2. 10. The method of claim 1, wherein response to anti-CD20 therapy constitutes survival for three or more years following the start of the therapy.
 11. The method of claim 1, wherein the control set of samples comprises a representative number of patients that exhibit response to anti-CD20 therapy and a representative number of patients that exhibit no response or poor response to anti-CD20 therapy.
 12. The method of claim 1, wherein determining whether the patient is likely to exhibit response to anti-CD20 therapy is made using statistical methods.
 13. (canceled)
 14. (canceled)
 15. A method of treatment of a diffuse large B-cell lymphoma (DLBCL) patient, comprising: (a) obtaining a sample from the patient; (b) detecting in the sample the expression of one or more genes selected from U2AF1, RNF2, ZC3H7B, CCNI, PSMD11, ABLIM1, GYG2, CAMK2G, NF1, gene coding for cDNA clone EUROIMAGE 826033 (NCBI Accession No. BE858194), TRIM66, FBXO21, MKNK2, FYTTD1, GET4, CABLES2, FMNL3, C22orf27, TBCEL, ZNF707, the gene corresponding to NCBI Accession No. AI763196, CSTB, BAHCC1, LOC723809, NPIP, the gene coding for CDNA FLJ40566 fis (NCBI Accession No. AL576118), LOC730367, RXFP2, MGC24125, the gene coding for mRNA, differentially expressed in malignant melanoma, clone MM A2 (NCBI Accession No. AJ293390), PTP4A1, OSBP, HNRNPD, AHCYL1, BMP2K, IDS, SERPINB9, CSRP2BP, CD58, HERC4, MAGOH, TMBIM4, SFRS12, FAS, ZNF333, the gene corresponding to NCBI Accession No. AI127295, USP6, CHD6, LOC653160, the gene corresponding to NCBI Accession No. R64696, SRPR, MIR21, ARHGAP18, the gene corresponding to NCBI Accession No. AW004885, CBFA2T2, TIMP3, CTBS, USP16, RASA1 and YJEFN3; (c) determining whether the patient is likely to exhibit response to anti-CD20 therapy, based on the similarity between the expression of each gene measured in step (b) in the patient's sample and a set of control samples, where the expression of the corresponding genes has been determined; (d) administering rituximab if in step (d), the patient is determined to be likely to respond to anti-CD20 therapy.
 16. The method of claim 15, wherein in step (b), the expression of all the genes listed in Table 1 is detected.
 17. The method of claim 15, wherein in step (b), the expression of all the genes listed in Table 2 is detected.
 18. (canceled)
 19. (canceled)
 20. (canceled)
 21. A kit for predicting whether a patient with diffuse large B-cell lymphoma (DLBCL) is likely to exhibit response to anti-CD20 therapy comprising: (a) a set of nucleic acid probes for one or more genes selected from U2AF1, RNF2, ZC3H7B, CCNI, PSMD11, ABLIM1, GYG2, CAMK2G, NF1, gene coding for cDNA clone EUROIMAGE 826033 (NCBI Accession No. BE858194), TRIM66, FBXO21, MKNK2, FYTTD1, GET4, CABLES2, FMNL3, C22orf27, TBCEL, ZNF707, the gene corresponding to NCBI Accession No. AI763196, CSTB, BAHCC1, LOC723809, NPIP, the gene coding for CDNA FLJ40566 fis (NCBI Accession No. AL576118), LOC730367, RXFP2, MGC24125, the gene coding for mRNA, differentially expressed in malignant melanoma, clone MM A2 (NCBI Accession No. AJ293390), PTP4A1, OSBP, HNRNPD, AHCYL1, BMP2K, IDS, SERPINB9, CSRP2BP, CD58, HERC4, MAGOH, TMBIM4, SFRS12, FAS, ZNF333, the gene corresponding to NCBI Accession No. AI127295, USP6, CHD6, LOC653160, the gene corresponding to NCBI Accession No. R64696, SRPR, MIR21, ARHGAP18, the gene corresponding to NCBI Accession No. AW004885, CBFA2T2, TIMP3, CTBS, USP16, RASA1 and YJEFN3; (b) reagents necessary for detecting hybridization of the nucleic acid probes.
 22. The kit of claim 21, comprising nucleic acid probes for the genes listed in Table
 1. 23. The kit of claim 21, comprising nucleic acid probes for the genes listed in Table
 2. 24. The kit of claim 21, comprising antibodies for proteins expressed from the genes listed in Tables 1 and
 2. 25. The kit of claim 21, further including a control set of samples, comprising a samples from a representative number of patients that exhibit response to anti-CD20 therapy and samples from a representative number of patients that exhibit no response or poor response to anti-CD20 therapy.
 26. The kit of claim 25, wherein the anti-CD20 therapy is treatment with rituximab. 